Ruggedized Apparatus for Analysis of Nucleic Acid and Proteins

ABSTRACT

The invention provides methods and systems for ruggedizing a nucleic acid analyzing apparatus. The ruggedized apparatus can be used reliably and effectively in uncontrolled environments, such as, for example at a crime scene to collect and analyze forensic data, as well as in semi-controlled environments, such as, for example at a point of care location.

RELATED APPLICATIONS

This application is a continuation of co-pending application Ser. No.13/482,765, filed on May 29, 2012, which is a continuation ofapplication Ser. No. 12/396,110, now issued as U.S. Pat. No. 8,173,417,which is a continuation of application Ser. No. 11/132,712, now issuedas U.S. Pat. No. 8,206,974, the entire disclosures of each of theseapplications and patents are incorporated herein by reference in theirentireties. All of these applications and patents are owned by theassignee of the instant application.

FIELD OF THE INVENTION

The invention generally relates to nucleic acid and/or protein analysisdevices, and more particularly to ruggedized devices with low powerconsumption requirements that can be used for nucleic acid and proteinsequencing or separation in uncontrolled or semi-controlled environmentsincluding mobile labs, physician's offices, hospital labs and otherhuman, veterinary or environmental clinical and/or testing labs andpoints of care locations.

BACKGROUND

The human genome includes stretches of DNA composed of short tandemrepeats (STRs). To date, hundreds of STR loci have been mapped in thehuman genome. The analysis of such STR loci and STRs is an importanttool for genetic linkage studies, forensics, and new clinicaldiagnostics. For example, forensic case work typically involvesseparation and analysis of multiple loci. Some tests use the 6 loci testknown as the “Second Generation Multiplex” (SGM), together withamelogenin (gender determining marker) and four additional loci,D351358; D19S433; D16S539 and D2S1338. Other commercially available kitssimultaneously amplify 15 tetranucleotide STR loci and the amelogeninmarker (See, e.g., AmpFlSTR Identifier PCR Amplification Kit). TheUnited States Federal Bureau of Investigation (FBI), European Network ofForensic Science Institutes (ENFSI) and Interpol generally recognizeresults from kits including at least the thirteen core STR locistandardized under the Combined DNA Index System (CODIS): CSF1PO,D3S1358; D5S818; D7S820; D8S1179; D13S317; D16S539; D18S51; D21S11; vWA;FGA; THO1; and TPDX. For a general discussion, see Budowle, B. et al.,“CODIS and PCR Based Short Tandem Repeat Loci: Law Enforcement Tools,”Second European Symposium on Human Identification, 1998, pages 73-88,hereby incorporated by reference in its entirety.

Studies of the human genome also has revealed, and continues to reveal,the existence of specific mutations or polymorphisms. With increasingfrequency, these mutations or polymorphisms are being associated withmonogenetic and polygenetic diseases. As a result, the field ofmolecular diagnostics is growing and expanding. Molecular diagnostictesting uses polymorphic markers, such as, microsatellites and STRs, andthe determination of mutations associated with neoplastic and otherdiseases. For example, the presence of certain viral infections, such asherpes simplex virus (HSV), cytomeglia virus (CMV) and humanimmunodeficiency virus (HIV) have been diagnosed using amplified andseparated DNA fragments. Certain types of cancer diagnosis also iscarried out using separation of amplified DNA fragments. Specifically,the diagnosis of B and T cell lymphomas fall into this category. Whencancer occurs, a single cell having a single form of rearranged DNAgrows at an elevated rate, leading to the predominance of that form ofthe gene. Separation and identification of the mutated gene can becarried out using conventional or microchip devices. For a discussion ofthe application of sequencing and separation methods and apparatus tomolecular diagnostics, see generally, “Use of Capillary Electrophoresisfor High Throughput Screening in Biomedical Applications, A Minireview,”by Bosserhoff et al. in Combinational Chemistry & High ThroughputScreening, 2000, issue 3, pages 455-66 and “Exploiting SensitiveLaser-Induced Fluorescence Detection on Electrophoretic Microchips forExecuting Rapid Clinical Diagnostics,” by Ferrance et al. inLuminescence, 2001, issue 16, pages 79-88, the disclosures of which arehereby incorporated by reference in their entirety.

A typical STR locus is less than 400 base pairs in length, and includessingle repetitive units that are two to seven base pairs in length. STRscan define alleles which are highly polymorphic due to large variationsbetween individuals in the number of repeats. For example, four loci inthe human genome CSF1PO, TPDX, THO1, and vWA (abbreviated CTTv) arecharacterized by an STR allele that differs in the number of repeats.Two repeating units are found at these loci: AATG for TPDX and THO1, andAGAT for CSF1PO and vWA.

In general, STR analysis involves generating an STR profile from a DNAsample, and comparing the generated STR profile with other STR profiles.Generating an STR profile typically involves amplifying an STR locususing PCR or another amplification method, dying or tagging STRs withina DNA sample, separating the tagged STRs within the sample usingelectrophoresis (applying an electric field), and recording the taggedSTRs using a laser or other fluorescence excitation device and agalvanometer or other device to direct the fluorescence excitationdevice towards a sample and then to a light detector.

One procedure for generating an STR profile uses an elongated gel plate(or slab gel) that is approximately 35 cm long. In general, this process(hereinafter referred to as “the gel plate process”) involves depositinga tagged nucleic acid sample (most often DNA, but as one skilled in theart will appreciate, RNA may also be used for some applications) on anarea of the gel plate, separating the STRs within the tagged DNA sampleon the gel plate using electrophoresis, and scanning the gel plate witha detector to record the tagged STRs. Typically, the gel plate processrequires two to three hours to complete.

Another procedure for generating an STR profile uses a capillary that is50 to 75 microns in diameter. This process (hereinafter referred to as“the capillary process”) generally involves electrokinetically injectinga tagged DNA sample at one end of a capillary, and drawing the samplethrough the capillary using electrophoresis to separate the STRs. Alaser beam is used to excite the tagged STRs within the sample to causethe tagged STRs to fluoresce. The fluorescence emitted by the STRs isdetected by scanning a portion of the capillary with a fluorescenceexcitation device, such as, for example a laser.

Typically, STR separation is faster in the capillary process than in thegel plate process. In general, an increase in electrophoresis currentresults in an increase in STR separation speed, and a higherelectrophoresis current typically can be applied to the capillary thanto the gel plate because the capillary more easily dissipates heat(caused by the current) that would otherwise skew the separationresults. A typical capillary process requires between 10 minutes and onehour to complete.

However, controlling temperature is a critical factor related to theprecision of capillary-based DNA separation devices. It illustrates whyprior art devices are not suitable for rugged, uncontrolled orsemi-controlled environments or applications. Prior art devices utilizean array of sixteen capillaries that are injected and run simultaneouslyat the same temperature, so intra-run precision can be expected to behigh, and data sized relative to an allelic ladder within the run can beexpected to be reliable. However, inter-run precision appears to bedependent upon temperature fluctuations. Whenever the temperaturechanges from run to run, the unknown fragments may not be able to besized by an allelic ladder in a different run. Fragments lying outsideof the bin may be called “off-ladder” alleles or mistyped by fallinginto an adjacent bin. As a result, samples analyzed using this type ofdevice may be mischaracterized, thereby significantly decreasing thequality of results obtained.

Another procedure for generating an STR profile uses a microfluidic chipprocess. Microfluidics technology is a term generally used to describesystems fabricated using semiconductor manufacturing techniques tocreate structures that can manipulate tiny volumes (microliter,nanoliter, or picoliters) of liquid, replacing macroscale analyticalchemistry equipment with devices that could be hundreds or thousandstimes smaller and more efficient. A microfluidic device (chip) isgenerally characterized by the presence of channels with at least onedimension less than 1 millimeter. Similarly, a microchannel is a channelwith at least one dimension that is less than about 1 millimeter.Microfluidic chips offer at least two major advantages as compared toconventional devices. First, the volume of sample and reagents requiredwithin these channels is small, allowing minimal sample sizes (generallya few nanoliters) and reducing reagent costs. Second, a systemcontaining such channels and similarly sized electrical or mechanicaldevices allows a wide array of complex sample manipulations to beperformed within a small volume. Finally, a system containing such smallstructures can be highly multiplexed to allow for simultaneousprocessing of multiple samples and therefore high throughput operation.

Chips generally are composed of durable transparent glass. A typicalchip consists of one or more channels fabricated within a planarsubstrate with access points for samples to be introduced into thechannel. In one embodiment, a channel can consist of a long arm(sometimes referred to as the “long channel”) and short arm (sometimesreferred to as the “short channel”). An individual STR separation can beperformed at each channel. The short arm intersects the long channelnear one end of the long arm and at an angle. In some chips, the shortarm includes a jog where it intersects the long channel such thatportions of the short channel are parallel but not co-linear. Typically,a chip is formed using photolithography and chemical etching techniquesto produce channel structures in fused silica wafers. These etchedchannel structures are bonded to an unetched fused silica wafer to forma complete channel structure.

An STR separation process that uses a chip generally involves orientingthe microchip so that it and the channels within lie horizontally (i.e.,perpendicular to the direction of gravity) and depositing a sample oftagged DNA over a hole in the upper surface of the microchip thatconnects with one end of the short channel of a channel pair. Next, thetagged DNA sample is drawn horizontally through the short channel usingelectrophoresis such that STRs within the sample are partially separatedalong the short channel. Then, a portion of the sample at theintersection of the long and short channels is further separated alongthe long channel using electrophoresis. A laser excites the tagged STRwhich fluoresces. The fluorescences emitted by the STRs is detected andrecorded in a manner similar to that of the gel plate and capillaryprocesses.

Conventional high-speed DNA genotyping using a chip is described in anarticle entitled “High-Speed DNA Genotyping Using MicrofabricatedCapillary Array Electrophoresis Chips,” Analytical Chemistry, Vol. 69,No. 11, Jun. 1, 1997 on pages 2181 through 2186, the teachings of whichare hereby incorporated by reference in their entirety. Ultra-high-speedDNA sequencing using capillary electrophoresis chips is described in anarticle entitled “Ultra-High-Speed DNA Sequencing Using CapillaryElectrophoresis Chips,” Analytical Chemistry, Vol. 67, No. 20, Oct. 15,1995 on pages 3676 through 3680, the teachings of which are herebyincorporated by reference in their entirety.

U.S. Pat. No. 6,207,031 issued to Adourian et al., the teachings ofwhich is hereby incorporated by reference in its entirety, describes anautomated separation device useful in allelic profiling assays. Theseparation device described in U.S. Pat. No. 6,207,031 includes amicrofabricated channel device having a channel of sufficient dimensionsin cross-section and length to permit a sample to be analyzed rapidly.Specifically, the microfabricated channel is filled with a replaceablepolyacrylamide matrix operated under denaturing conditions and afluorescently labeled STR ladder is used as an internal standard forallele identification. Samples analyzed by the assay method can beprepared by standard procedures and only small volumes of assay arerequired per analysis. This device is capable of repetitive operationand is suitable for automated high-speed and high-throughputapplications.

While the separation devices described in U.S. Pat. No. 6,207,031 areefficient at separating a large number of samples in a relatively shortamount of time, the device of U.S. Pat. No. 6,207,031 needs to belocated in a laboratory setting under a controlled environment. As aresult, in field use, such as, for example, use of the above-describeddevices at a point of care location, such as a doctor's office or at anemergency disaster site is impractical due to the size, energyrequirements, and operational (i.e., minimal vibrational impact onsensitive optical equipment) constraints of these devices. In addition,these devices also have high power consumption that limits the utilityof these devices in field use.

Thus, there is an unmet need in the industry to ruggedize sequencing andseparation devices so that reliable, timely measurements can be obtainedin the field or in a non-controlled environment. Further, there is anindustry need to provide a commercially available suitably ruggedizedsequencing and/or separation device using microfluidics.

SUMMARY OF THE INVENTION

In general, the present invention relates to miniaturized, fast, highlyruggedized nucleic acid sequencing and separation devices. Such deviceshave uses in physician's offices, hospital laboratories, and otherhuman, veterinary and environmental clinical and/or testing laboratoriesand other point of care locations. The device of the present inventionis suitable for use by unskilled or semi-skilled operators and inenvironmental locations that are controlled or semi-controlled. Thedevices of this invention can be expected to deliver significantperformance improvements over prior art machines. The devices of thepresent invention offer the compelling advantages of dramaticallyreducing the amount of sample and reagent and enabling unique microscalechemistries not possible with macroscale instrumentation, while beingable to operate in uncontrolled, semi-controlled, such as, for example aphysician's office, or even hostile environments, such as, for example,mobile forensic crime labs, labs or diagnostic stations in harsh orsemi-harsh physical locations or environments.

In addition, the devices described in accordance with the presentinvention can include a novel system that maintains the temperature ofthe capillary array or its equivalent in a very precise temperaturerange (e.g., within ±1 degree C.), thereby minimizing run-to-runvariation due to changes in temperature in the external environment. Asa result, the number of “off-ladder” alleles generated during separationis reduced and thus, the quality of the results obtained is increasedover prior art devices. Furthermore, embodiments of the inventiondescribed herein are implemented using low power levels, thereby makingmachines and devices in accordance with the present invention suitablefor portable and/or in field use.

The present invention also relates to the support of light detecting andemitting elements including mirrors, lasers, scanners and the like for aDNA separation/sequencing device. The nucleic acid and protein analysisdevices of the present invention incorporate specialized laser opticsand sensitive optical scanning devices that reproducibly detectmicrofluidic lanes for satisfactory performance. Since each of thecomponents of a light emission and detection system (e.g., a laser,mirrors, scanners, filters, light detectors) controls some portion ofthe laser beam's path, any movement or dislocation of a component maycause a disturbance in data collection. As a result of thesedisturbances, data flow can be interrupted or corrupted, therebyproducing incorrect results. Prior art devices using capillary systemsalso use laser based light emission and detection systems, andtherefore, may be adapted for ruggedization using the inventionsdescribed herein. The device of the present invention includes a highlyruggedized laser optic and detector system to reproducibly detectmicrofluidic lanes that minimizes the amount of noise produced in thedetected signal as a result of vibration and/or shock. In addition, incertain embodiments, the invention relates to a device that requiresless energy than conventional laboratory sequencing devices forelectrophoresis and analysis. As a result, the device of the presentinvention can be used reliably in the field while obtaining highresolution results. The nucleic acid sequencing and separation devicesof the prior art also use specialized laser optics and detection systemsin capillary lanes. One skilled in the art could readily adapt the laseroptics and detection systems generally used in capillary systems to beruggedized according to the teachings of this invention.

In general, the term “biomolecular analyte” as used herein refers toboth non-synthetic and synthetic nucleic acids (e.g., DNA and RNA) andportions thereof, and biological proteins. Described herein arepractical ultra-fast techniques for allelic profiling of suchbiomolecular analyte in the field. In particular, the techniques involveusing a microfluidic electrophoresis device to analyze short tandemrepeats (STRs) within a nucleic acid or protein sample, or to otherwiseseparate DNA molecules to allow determination of a nucleic acidsequence. Moreover, as one skilled in the art will appreciate, thesample may be modified or “tagged” to include something that can bedetected by a detection device. Tagging may be accomplished by anyapplicable method, including without limitation, incorporating a dye,chromofore, fluorophore, calorimetric adduct or radioactive adduct intothe sample. An assay method of the present invention has made itpossible to rapidly achieve baseline-resolved electrophoreticseparations of single-locus STR samples. In one embodiment, analysis ofsamples (e.g., PCR samples) containing loci defined or characterized byan STR which differs in the number or repeats is performed rapidly usingthe allelic profiling assay described herein. For example, analyses ofPCR samples containing four to five loci can be performed in less thanabout thirty minutes.

Also described herein is a separation device (or test module) useful inan allelic profiling assay of the present invention. The separationdevice includes a microchannel device having a channel of sufficientdimensions in cross-section and length to permit a sample to be analyzedrapidly. In one embodiment, the separation device consists of amicrochannel having a cross-sectional area of from about 300 to about16,000 square microns in cross-section and about 100 to 500 mm inlength. In certain embodiments the microchannel has a preferredcross-sectional area of about 2,500 square microns and a length of 180mm. A fluorescently labeled STR ladder is used as an internal standardfor allele identification. Samples analyzed by the assay method can beprepared by standard procedures and only small volumes (e.g., 4microliters or less) are required per analysis.

In addition, the devices of this invention can be used in sequencing.Although a large amount of sequencing of the human and other genomes hasbeen completed, there is still good reason to sequence small parts ofgenomes, especially in a semi-controlled or uncontrolled environment.For example, over 1,000 different mutations have been found in patientswith cystic fibrosis. Each of these mutations occurs in a huge gene thatencodes a protein of 1,480 amino acids known as the cystic fibrosistransmembrane conductance regulator (CFTR). The gene encompasses over6,000 nucleotides spread over 27 exons on chromosome 7. Defects in theprotein cause various symptoms of the disease. No single mutation isresponsible for all cases of cystic fibrosis. People with the diseaseinherit two mutant genes, but the specific mutation need not be thesame. Therefore, the ability to identify the specific mutation has somepredictive value. As a further example, the AIDS virus mutates veryquickly and a treating physician has a need to be able to sequence themutated virus at a point of care location so a patient can be sent homewith the appropriate therapy to combat the mutated virus at each stageof the course of the disease. In yet another application, an oncologicsurgeon making an intra-operative diagnosis in a cancer patient has aneed for the ability to receive sequence information at the point ofcare location.

In general, in one aspect, the invention features a ruggedized apparatusfor analyzing a sample of biomolecular analyte. The ruggedized apparatusincludes a holder, an electrophoresis device, a low powered light source(i.e., a light source drawing less than about 10 amps at 240 volts), alight detector, and a plurality of optical devices. The holder of theruggedized apparatus supports a transparent test module having at leastone channel disposed with the transparent test module. Theelectrophoresis device is connected to the holder and provides energy tothe transparent test module. The light source emits a light beam that iscapable of exciting the sample of biomolecular analyte. The plurality ofoptical devices mounted within the apparatus transmit the light beamfrom the light source to the transparent test module to excite thefluorescently tagged biomolecular analyte sample. These optical devicesalso collect fluorescence from the biomolecular analyte sample andtransmit this fluorescence to the light detector.

In one embodiment of the invention, the ruggedized apparatus furtherincludes a portable power supply including a maximum power consumptionof 1.5 kVA or less to provide electrical energy to the ruggedizedapparatus. In another embodiment of the invention, the electrophoresisdevice includes a heater to provide thermal energy to the transparenttest module and a plurality of pairs of electrodes to provide electricalenergy to the plurality of separate microchannels. In anotherembodiment, the apparatus includes a heater to provide thermal energy tothe transparent test module and a plurality of pairs of electrodes toprovide electrical energy to the at least one channel.

The plurality of optical devices can be rigidly mounted to a base platedisposed within the apparatus. The light source of the apparatus can bea low power laser, such as a solid state laser or any other suitablelaser. The at least one channel of the transparent test module can be amicrochannel or a plurality of channels.

In another aspect, the invention features an apparatus for analyzing asample of biomolecular analyte. The apparatus includes a holder forsupporting a transparent test module having at least one microfluidicchannel disposed therein. An electrophoresis device is connected to theholder and provides energy to the transparent test module. A low poweredlight source emits a light beam that excites fluorescence in the sampleof biomolecular analyte. A plurality of optical devices mounted withinthe apparatus transmit the light beam emitted by the light source to thetransparent test module and from the transparent test module to a lightdetector.

In one embodiment, the plurality of optical devices are rigidly mountedto a base plate disposed within the apparatus. In another embodiment,the electrophoresis device comprises a heater to provide thermal energyto the transparent test module and a pair of electrodes to provideelectrical energy to the at least one microfluidic channel. In someembodiments, the transparent test module includes a plurality ofmicrofluidic channels. In certain embodiments, the low powered lightsource includes a low power laser. In some embodiments, the apparatusfurther includes a portable energy source to provide energy to theapparatus. The portable energy source can have a maximum powerconsumption of less than about 1.5 kVA.

In another aspect, the invention features an apparatus for processing asample of biomolecular analyte. The apparatus includes a holder forsupporting a transparent test module having at least one channeldisposed therein. An electrophoresis device is connected to the holderand provides energy to the transparent test module. A light source foremitting a light beam excites fluorescence in the sample of biomolecularanalyte. A plurality of optical devices are rigidly mounted to a baseplate formed from a single piece of material. The optical devicesdisposed on the base plate transmit the light beam emitted by the lightsource to the transparent test module and from the transparent testmodule to a light detector. In one embodiment, the base plate issupported by a frame including a damping device to reduce transmissionof vibrations generated below the frame to the base plate. The baseplate can include a plurality of securing elements for limiting therotation movement of the plurality of optical devices on the base plate.

In one embodiment, the holder for supporting the transparent test modulecan include a first member with a pair of electrodes disposed toelectrically connect with openings disposed on a transparent testmodule. A second member includes an automated locking feature to reducelateral motion of the transparent test module when the transparent testmodule is positioned between the first member and the second member.

In one embodiment, the light source of the apparatus is a solid statelaser, a low power laser, or any other suitable laser. The lightdetector of the apparatus can include at least five photomultipliers.

In another aspect, the invention features a transparent test module foranalyzing a sample of biomolecular analyte. The transparent test moduleincludes at least one fluid channel disposed within a member formed of atransparent material. Each of the at least one fluid channel includes aseparation portion, a waste arm portion, a first sample arm portion, anda second sample arm portion. In one embodiment, the first sample armportion is adapted to receive a biasing solution and the second samplearm portion is adapted to receive the sample of biomolecular analyte. Inanother embodiment, the at least one fluid channel includes amicrofluidic channel. The transparent test module can be formed fromglass, from a polymer, a co-polymer, or any combination of these.

In another aspect, the invention features a method of determining centerchannel locations in a transparent test module including a plurality ofseparate fluid channels disposed therein. The method includes: providinga transparent test module including the plurality of separate fluidchannels, each of the separate fluid channels having a front endposition, a center channel position, and a known channel width; scanningover a portion of the transparent test module including each of theplurality of separate fluid channels with a light beam to generate areflected light beam; collecting the reflected light beam with a lightdetector to generate a waveform of intensity through the portion of thetransparent test module including each of the plurality of separatefluid channels; eliminating peaks determined not to be associated withthe plurality of separate fluid channels; identifying a location withinthe waveform of the front end position for each of the plurality ofseparate fluid channels; identifying all remaining peaks along thewaveform within a first distance extending from each of the identifiedfront end positions; and determining the center channel positions ofeach of the plurality of separate fluid channels from an average oflocations of all of the remaining peaks within the first distance fromeach of the identified front end positions.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the invention will beapparent from the following more particular description of preferredembodiments of the invention, as illustrated in the accompanyingdrawings in which like reference characters refer to the same partsthroughout the different views. The drawings are not necessarily toscale, emphasis instead being placed upon illustrating the principles ofthe invention.

FIG. 1A is a perspective view of a portable system for processingbiomolecular analyte according to the invention. A sample holder of theportable system is shown in an open position.

FIG. 1B is another perspective view of the portable system of FIG. 1A.The sample holder of the portable system is shown in the closedposition.

FIG. 1C is a side view of the portable system of FIG. 1A. A portion of acover has been removed so that a portion of the interior of the portablesystem is visible.

FIG. 2 is a schematic view of a laser excitation and fluorescencedetection system used within the portable processing system of thepresent invention.

FIG. 3A is a top view of a test module that is usable in the systems ofFIGS. 1A and 1B.

FIG. 3B is another top view of a test module that is usable in thesystems of FIGS. 1A and 1B. The test module shown in this figureincludes an attached sample board for holding samples and fluids to beinjected into the test holder.

FIG. 4 is a schematic view of a channel usable in the test module ofFIG. 3.

FIG. 5 is a schematic view of another channel usable in the presentinvention.

FIG. 6 is a schematic view of an embodiment of a first biasing conditionapplied to a test module used in the present invention duringelectrophoresis.

FIG. 7 is a schematic view of an embodiment of a second biasingcondition applied to the test module during electrophoresis.

FIG. 8 is a schematic view of an embodiment of a third biasing conditionapplied to the test module during electrophoresis.

FIG. 9 is a schematic view of an embodiment of a fourth biasingcondition applied to the test module during electrophoresis.

FIG. 10 is a schematic view of an embodiment of a fifth biasingcondition applied to the test module during electrophoresis.

FIG. 11 is a schematic view of an embodiment of a sixth biasingcondition applied to the test module during electrophoresis.

FIG. 12 is a schematic view of an embodiment of a seventh biasingcondition applied to the test module during electrophoresis.

FIG. 13 is a schematic view of an embodiment of a test module disposedwithin a portion of a holder.

FIG. 14 is an illustration of laser beam paths through a test module.

FIG. 15 is a graph of light intensity versus position across a channelof a test module.

FIGS. 16 A-D are graphs of raw data collected from a sample including acommercially available allelic ladder and a size standard.

FIGS. 17 A-D are graphs of corrected data of the raw data shown in FIGS.16 A-D.

FIGS. 18 A-D are graphs of raw data collected from a sample including aDNA template from an HIV amplicon with primer.

FIGS. 19 A-D are graphs of corrected and base called data the raw datashown in FIGS. 18 A-D.

FIGS. 20A-20C are sequence listings of a known HIV amplicon (SEQ IDNO:1), unedited data generated from a nucleic acid analyzing device ofthe present invention (SEQ ID NO: 2), and edited data generated from thenucleic acid analyzing device of the present invention (SEQ ID NO: 3),respectively. For formatting purposes, FIG. 20A is spaced over two pagesFIG. 20A-1 and FIG. 20A-2.

DETAILED DESCRIPTION

There is a compelling need for a ruggedized DNA separation device thatcan analyze samples reliably and quickly in the field (i.e.,non-laboratory use). Traditional approaches to ruggedizing DNAseparation devices have included adding bulky cushioning and/orstructural support members. These approaches have not met industry'sneeds for a number of reasons including: the non-portability of thedevices due to the extra weight of the cushioning and/or structuralsupport members, the unreliability of device results due to an increasein background noise generated by uncontrolled vibrational forces actingupon sensitive equipment, and the inherent high power consumption ratesneeded for conventional devices.

In contrast to traditional approaches, an embodiment of the invention isdirected to a ruggedized DNA separation/sequencing device (e.g., adevice that can be mounted in a mobile forensic unit) used to analyzebiomolecular analyte. In general, the invention provides fast, reliableseparation and sequencing analysis out in the field. Referring to FIGS.1A, 1B, and 2, the ruggedized separation device 10 includes a sampleholder 20, an electrophoresis assembly 30 connected to the holder 20,and a fluorescence excitation and detection system 40 (see FIG. 2)located underneath the sample holder 20 and covered by protective cover50 (see FIG. 1A). The fluorescence excitation and detection system 40includes an opening 42 within the holder 20 so that an energy sourcethat induces fluorescence (e.g., a laser beam) and the resulting inducedfluorescence can pass between the holder 20 and the fluorescenceexcitation and detection system 40.

Another embodiment of the invention is directed to noncontrolled orsemi-controlled environments such as police department offices, mobileforensic labs, physician's offices, hospital laboratories, clinicallaboratories and other points of care locations. In such settingsnon-skilled or semi-skilled operators must be able to operate andmaintain the devices at low cost and with little to no training orexperience required. In addition, these settings may be in environmentallocations where ambient conditions vary significantly. The devices mustbe able to withstand temperature and other environmental fluctuationswithout affecting results. One aspect of this is the ability to sustainshock and vibration without the need for realignment orre-initialization. Furthermore, in many of these environments there is aneed for rapid and accurate results while the patient is still with auser of the device (e.g., a treating physician, a police detective, aforensic scientist, or other user). For example, devices in accordancewith the present invention can be used by an infectious diseasephysician during an examination to select an appropriate antibodytherapy to give to a sick patient, or by a oncologist surgeon to make anintra-operative diagnosis in a cancer patient.

The sample holder 20 receives and supports a test module 55 including aplurality of separate microchannels. Samples of biomolecular analyte areinjected into the microchannels and then the test module 55 is placedwithin the holder 20 for analysis. After inserting the test module 55into the holder 20, a user places the holder 20 in a closed position(see FIG. 1B) so that the electrophoresis assembly 30 makes contact withthe test module 55. With the holder 20 in the closed position, the useractivates the electrophoresis assembly 30 to apply a voltage to the testmodule 55 so that components consisting of biomolecular analyte (e.g.,STRs) separate.

The size of each component within or attached to device 10 can beminiaturized by any one or a combination of the following factors:changing the type of energy source used to induced fluorescence, varyingthe size of the chip, reducing power consumption needs, reducing thesize of the electronics and incorporating sample preparation into theunit. For example, a small aliquot of blood can be introduced into oneport of the device and routed to a sample preparation area for DNAextraction. The resulting DNA sample then may be manipulated andanalyzed by the microfluidic chip as described elsewhere in thisspecification.

The fluorescence excitation and detection system 40 excites thecomponents separated by electrophoresis of a DNA sample (e.g., STRs) byscanning an energy source (e.g., a laser beam) through a portion of eachof the microchannels while collecting and transmitting the inducedfluorescence from the biomolecular analyte to one or more lightdetectors for recordation and ultimately analysis. In one embodiment,the fluorescence excitation and detection assembly 40 includes a laser60, a scanner 62, one or more light detectors 64, and various mirrors68, filters 70 (e.g., band-pass filters, dichotic filters), and lenses72 for transmitting a laser beam emitted from the laser 60 throughopening 42 to the test module 55 and back to the light detectors 64. Thescanner 62 moves the incoming laser beam to various scanning positionsrelative to the test module 55. Specifically, the scanner 62 moves thelaser beam to a pertinent portion of each microchannel within the testmodule 55 to detect respective separate components. The one or morelight detectors (e.g., photomultiplier tubes, photodiodes, CCD camerasor linear array detectors) 64 collect data (e.g., the fluorescentDNA/STRs signal) from the test module 55 and provide the dataelectronically through a cable attached to port 75 to a data acquisitionand storage system located outside the protective cover 50. In oneembodiment, the data acquisition and storage system can include aruggedized computer available from Option Industrial Computers(Baudreuil-Dorion, Quebec, Canada).

Uncontrolled vibrations that interact with the fluorescence excitationand detection system 40 can be detrimental to data collection andultimately lead to problems with obtaining reliable results. Theseproblems can be exacerbated when a DNA separation/sequencing device isused in an uncontrolled environment, such as when the device istransported and used at a crime scene (i.e., field use). To reduce theeffects of environmental vibration, the present invention includes oneor more of the following various elements. These elements each have aminimal impact on the overall weight or bulk of the device and thus,allow for the device of the present invention to be easily transported(i.e., portable). For example, in one embodiment, the fluorescenceexcitation and detection system 40 resides on a plate 80 formed from asingle piece of material (i.e., unitary construction). That is, thelaser 60, scanner 62, the one or more light detectors 64, the mirrors,68, filters 70, and the lenses 72 are secured to plate 80 made from asingle piece of material, such as, for example a single piece ofaluminum. As a result, all the components of the fluorescence excitationand detection system 40 have a common base that has no joints or otherintersections that could potentially transmit and or generate vibrationsto the fluorescence excitation and detection system 40. The componentsof the fluorescence excitation and detection system 40 are secured tothe single plate 80 with one or more fasteners commonly used in theoptics industry. In addition to using the fasteners, some embodiments ofthe invention also include securing elements for further reducing therotational movements of these components during use. Since each of thecomponents of the fluorescence excitation and detection system controlssome portion of the laser beam's path, any movement of the componentscan cause a disturbance in data collection. As a result of thesedisturbances, the data flow can be interrupted or corrupted, therebyproducing unreliable results. The securing elements, which can beattached to the plate 80 prior to the installation of the componentsthereover, extend vertically away from the plate 80 and fit intoopenings created in the components to limit the components' rotationalmovement thereof during use. For example, in one embodiment, thesecuring elements comprise dowel pins extending from the plate 80. Thecomponents of the fluorescence excitation and detection system 40 arepositioned on to the plate 80 such that apertures in the components fitsnuggly over the dowel pins to limit the rotation of the components. Tohold the components to the plate 80, fasteners, such as, for example,screws are used to connect the components to plate 80.

As a further example of how vibration and shock can be controlled in thepresent invention, some embodiments include one or more damping devicespositioned between the ground and the plate 80 supporting thefluorescence excitation and detection system 40. These damping devicesabsorb vibrational forces and reduce the transmission of vibrations tothe fluorescence excitation and detection system 40. For example, in theembodiment of the invention shown in FIGS. 1A, 1B, and 1C, the device 10includes a frame 82 that supports the plate 80. Disposed between theframe and the plate 80 are four damping coils 84 (two of which are inview in FIGS. 1A and 1B, four in FIG. 1C) that absorb shock and otherforces, thereby preventing or substantially reducing transmission ofvibration to the fluorescence excitation and detection system 40. Thedamping coils 84 in the embodiment shown in FIGS. 1A and 1B arepositioned to equally balance the weight of the device. That is, eachcoil 84 supports an equal amount of weight. In other embodiments, thedamping coils 84 can be positioned symmetrically about the frame 82 orcan be positioned as desired. Moreover, more or less than four dampingdevices can be used to absorb shock. Examples of suitable dampingdevices for controlling shock and vibration include coils of wire ropeisolators (e.g., stainless steel wire rope available from Enidine ofOrchard Park, N.Y.) or metal springs having a large stiffnesscoefficient, pneumatic or hydraulic shock absorbing mounts, andrubberized shock absorbing mounts.

In some embodiments, automated locking features are used to reducemotion of the test module 55 during analysis. For example, in certainembodiments of the present invention, the holder 20 can further includenon-moveable test module stops 86 and automated positioning bumpers 88.The automated positioning bumpers 88 move towards the non-moveable stops86 as the top portion of the holder 20 is closed. Thus, the automatedpositioning bumpers 88 guide and push an inserted test module 55 upagainst the non-moveable test module stops 86. As a result, the testmodule 55 is positioned snuggly between the test module stops 86 and theautomated positioning bumpers 88, thereby locking the test module inplace and reducing lateral movements of the test module during analysis.

All of the features described above can be used alone or in combinationto provide shock absorption and prevent vibration transmission to thefluorescence excitation and detection system 40 without adding excessweight to destroy ruggedized nature and/or the small, miniaturized sizeof the device 10.

Other features of the present invention also contribute to theruggedized nature of the device 10. For example, the laser 60 usedwithin the fluorescence excitation and detection system 40 can beselected to emit a large amount of power while using little energy. As aresult, reliable measurements can be made and detected by using laser60, while not requiring a large amount of power from an external energysource connected to the device 10 through port 77. In general,conventional, stationary devices, such as the type used in laboratories,use gas lasers (e.g., helium-neon lasers). These gas lasers require alarge amount of energy to lase properly. For example, a typical gaslaser draws about 20 to 30 amps at 240 volts (i.e., requiring 7,200 W ofpower and having a maximum power consumption of about 2.5 kVA orgreater). Thus, a portable energy source connected to this type of laserwould have to be of a size large enough to support this power usage. Ingeneral, solid state lasers require less energy and are more efficientthan their gas phase laser counterparts, and thus contribute to theportability and ruggedized nature of the present invention. An exampleof a laser which requires limited power for proper operation and issuitable for use with the present invention is a solid state laser, suchas, for example, a diode pumped solid state laser. Typically, diodepumped solid state lasers use less than 2 amps at 240 volts (i.e., apower use of 480 W and having a maximum power consumption of less than1.5 kVA and in some cases, less than 0.5 kVA). In some embodiments,lasers or other light sources that draw less than 10 amps at 240 voltscan be used without reducing the portability of device 10. For example,a light source that draws less than 10 amps at 240 volts uses less than3,000 W of power. These light sources can be powered by a small portableenergy source (e.g., a 6,000 W or less power supply; a 1.5 kVA or lessmaxium power consumption power supply), thereby preserving theminiaturized nature of the present invention. In one embodiment of thepresent invention, the light source comprises a solid state, such as aSapphire 488 HP (Santa Clara, Calif.) solid state laser, which has amaxium power consumption of 0.75 kVA. In addition, the Sapphire solidstate laser can withstand 7 g of lateral and 15 g of vertical shock. Asone skilled in the art will appreciate, any laser with the desiredoptical properties can be used within the teachings of the presentinvention.

A thin thermofoil heater 90 disposed within the holder 20 alsocontributes to the portability of device. The heater 90 is situatedwithin holder 20 to be in thermal contact with an inserted test module55. This design allows the dimensions of the heater 90 to be small andequivalently sized with the test module 55. The heater 90, which may beactivated through a remote controller located outside of the device 10,can be used to apply thermal energy to the test module 55. The heatapplied to the test module 55 aids in separation of the biomolecularanalyte (i.e., the heater 90 is part of the electrophoresis device 30).The use of the small thermofoil heater 90 in combination with the testmodule 55 (e.g., a planar microfluidic chip) allows for efficient heattransfer between the heater 90 and the test module 55. As a result, alarge reduction in the amount of electric energy or power consumptionrequired to heat the test module and to maintain improved temperaturecontrol is achieved. In conventional capillary electrophoresis systems,heating and temperature control of the capillaries is achieved byplacing the capillaries in an oven. The large volumes of the ovens andinefficient heat transfer between the oven heater elements and thecapillaries results in the need for a large amount of energy, whichcould be potentially draining on a portable energy source and/or requirea larger power supply, thereby decreasing the portability of the nucleicacid separation/sequencing device. As a result, the present device canbe serviced by an attached, small, portable energy source (e.g., 6,000 Wor less and/or a 1.5 kVA or less maximum power consumption) forsignificant time periods, thereby increasing the portability of thepresent invention.

In addition to aiding in separation of the biomolecular analyte, theheater 90 also contributes to obtaining high resolution results. Forexample, the intimate contact between the heater 90 and the test module55 provides efficient heat transfer there between. As a result, theheater can maintain the temperature of the test module 55 within ±1degree C. of a desired temperature setting, thereby minimizing anydetrimental environmental temperature effects.

Separation or electrophoresis of the biomolecular analyte occurs withinthe microchannels in the test module 55. In general, the test module 55is made from a transparent material that allows at least a part of theenergy source inducing fluorescence (e.g., laser beam) from thefluorescence excitation and detection system 40 to transmit through thetest module 55 to interact with the sample located therein. Examples ofsuitable transparent materials include glasses (e.g., aluminosilicateglass, borosilicate glass, fused silica glass, and soda lime glass),single crystal alumina, and clear polymers or copolymers (e.g.,polymethyl methacrylate, uv treated polycarbonate, or cyclic olefincopolymer).

Referring to FIGS. 3A and 4, one or more microchannels 110 (16 channelsare shown in FIG. 3A, and one channel is shown in FIG. 4) each include asample arm 112, a waste arm 114, and a separation channel 116. Themicrochannels 110 can be manufactured on a transparent plate byutilizing standard photolithography and chemical etching procedures tomake channels having a depth of about 20 to 100 microns and a width ofabout 40 to 2,000 microns. In one embodiment, the preferred channeldepth is about 40 microns and the preferred channel width is 90 microns.As shown in FIG. 4, sample and waste arms 112 and 114 are offset fromone another at a distance of about 50 to 1,000 microns along a length ofthe separation channel 116. In one embodiment, the sample and waste arms112 and 114 are offset from one another at a distance of 500 micronsalong a length of the separation channel 116. Openings 118 (e.g., 118 a,118 b, 118 c, 118 d) to insert samples and remove waste as well as tomake electrical connections to the anode 100 and the cathode 102 arelaser drilled into the transparent plate. For example, in oneembodiment, a plate including 16 microchannels includes 16 sample holes,16 waste holes, 1 or more anode holes and 1 or more cathode holes. Inanother embodiment in which includes multiple test modules 55 (e.g., 10test modules each having 16 microchannels) formed on a singletransparent plate includes 160 sample holes, 160 waste holes, 160 anodeholes and 160 cathode holes. In still yet another embodiment, whichincludes two sample arms 112 a and 112 b per microchannel, a plate islaser drilled to include 32 sample holes, 16 waste holes, 1 or moreanode holes, and 1 or more cathode holes per test module 55. Thesemicrochannel structures are patterned and etched into a transparentplate by traditional semiconductor fabrication processes. Following thepatterning, ports for accessing the channels are formed by drillingprocesses, including laser drilling, abrasive jet drilling, andultrasonic drilling. The etched transparent plates are then cleaned withmultiple cleaning processes to remove debris and surface contamination.

To close and seal the channels 110 so that they can be filled withbiomolecular analyte, the etched transparent plate is thermally bondedto a blank or non-etched transparent plate. The bonding process isperformed in an oven in a two step process. The two step bonding processincludes positioning the two transparent plates (i.e., one etched andone non-etched) in the oven with the non-etch plate covering the etchedsurface of the etched plate so that a seal forms at all points includingin between the etched microchannels. The transparent plates are heatedto a temperature less than about 200 C for about 120 minutes. A force ofabout 0.5 to 10 pounds is uniformly applied to the plates throughout theheating process to promote the thermal bond. Upon completion of theabove low temperature heating process, the bonded transparent testmodule 55 is visually inspected. If there are no apparent cracks orother damage, the bonded transparent test module 55 is heated in a hightemperature oven at a temperature of about 735 degrees C. to completethe seal. The above thermal bonding process is further described inSemiconductor Wafer Boding: Science and Technology by Q.-Y. Tong and U.Gosele, published by Wiley Publishers, November 1998, which is herebyincorporated by reference in its entirety.

Referring to FIG. 3B, in certain embodiments, the test module 55includes a sample board 57 attached to a top surface including theopenings to the etched microchannels. The sample board 57 includes anumber of reservoirs 59 for holding samples and fluids used inelectrophoresis. In addition to serving as a holder for fluids, thesample board 57 also aids in providing proper alignment of the testmodule 55 in the holder 20. For example, the reservoirs 59 arepositioned within the sample board 57 to provide alignment andregistration of the reservoirs and the electrodes of the electrophoresisassembly 30 when the test module 55 is inserted into the holder 20.

Once the test module 55 is sealed, the internal surfaces of the channelsare treated to prevent electroosmosis and sample-wall interactions usinga slightly modified Hjerten protocol as described by Luba Mitnik et al.in Electropheresis 2002, volume 23, pages 719-26, hereby incorporated byreference in its entirety. The channels are filled with a sieving matrixmaterial and the openings leading the channels are filled with either abuffer solution or deionized water. The openings 118 are sealed and thetest module 55 is stored until it is needed for use (i.e., until thetest module is needed to hold a sample of biomolecular analyte foranalysis).

When one or more samples are ready to be analyzed the seal on the testmodule 55 is removed and the buffer solution or deionized water isremoved from each of the openings leading to channels 110 etched intothe test module 55. To condition the channels for use, the openings tothe channels 110 are flushed with deionized water three times. Each timethe water is removed by aspiration until dry. An electrophoresis gel orsieving matrix/material is injected into each of the channels with asyringe. As one skilled in the art will appreciate, a variety of sievingmaterials may be used. An example of a suitable sieving material is ahigh molecular weight linear polyacrylamide (LPA), such as, for example,a high molecular weigh 4% LPA commercially available from DakotaScientific (Sioux Falls, S.D.). Examples of other suitable sievingmaterials are described by Methal. et al., in “Polymeric Matrices forDNA Sequencing by Capillary Electrophoresis” in Electrophoresis 2000,Vol. 21, pages 4096-4111 and by Ruiz-Martinez in “DNA Sequencing byCapillary Electrophoresis with Replaceable Linear Polyacrylamide andLaser-Induced Fluorescence Detection,” in Anal. Chem., Vol. 65, pages2851-58, 1993, both disclosures of which are hereby incorporated byreference in their entirety.

The channel is filled with three times the volume of the channel worthof sieving gel from the anode opening 118 b first and then three timesthe volume of the channel worth of the sieving gel from the cathodeopening 118 a to coat the microchannel 110 completely with the gel andensure that all water has been removed. The openings 118 at the anode100 (i.e., opening 118 b), the cathode 102 (opening 118 a), and thewaste arm 114 (opening 118 c) are then filled with a buffer solution andthe opening 118 d of the sample arm 112 is filled with deionized water.

To complete the conditioning of the microchannels, the test module 55 isinserted into the holder 20 and the holder 20 is closed so that thecathode 102 and anode 100 connections on the holder 20 interface withthe cathode opening 118 a and anode opening 118 b on the test module 55.A first biasing configuration is applied to the test module 55 tocondition the channels in which a voltage of less than about 10 KV isapplied across the cathode 102 and anode 100 to move the sieving matrixdown the separation channel 116 from the cathode opening 118 a towardsthe anode opening 118 b. Then a second biasing configuration is appliedto the test module 55 to complete the conditioning. The second biasingconfiguration includes applying a voltage of less than 4 KV between thesample arm 112 and the waste arm 114 to move the ions from the samplearm opening 118 d into the waste arm opening 118 c. The test module 55is removed from the holder 20 and cleaned to remove all of the water andbuffer. Each of the ports is rinsed with deionized water at least threetimes and then the microchannels 110 are visually inspected undermagnification to check for the presence of bubbles. The test module 55is now ready for use. The conditioning processes are performed on thetest module at a temperature of between 40 to about 70 degrees C. Thatis the heater 90 warms the test module to a temperature of between 40 toabout 70 degrees C. during the conditioning process. In certainembodiments, the preferred temperature of operation is 50 degrees C.

An operator loads a sample of biomolecular analyate (including afluorescent dye to mark the STRs) into the test module 55 by injecting asample or a test control into one or more of the sample openings 118 dof the microchannels 110 through the sample board 57. The anode, cathodeand waste openings 118 a, 118 b, and 118 c are filled with the buffersolution. The test module 55 is placed back into the holder 20 and theanode 100 and cathode 102 are connected to the test module 55.

In another embodiment of the invention, one or more of the abovecleaning steps is eliminated in the conditioning process. Specifically,in certain embodiments of the invention (see FIG. 5), a test moduleincludes at least one microchannel 110 having a separation channel 116,a waste arm 114, and two sample arms 112 a and 112 b. As a result ofincluding the two sample arms 112 a and 112 b, cleaning processes usedto clean the test module 55 after conditioning is eliminated. That is,the microchannels are cleaned once, prior to adding the sieving matrix.In this embodiment, sample arm 112 a is filled with deionized water andsample arm 112 b is filled with biomolecular analyte including thefluorescent dye. The microchannel 110 is first conditioned by applyingthe first and second biasing configurations. Then, the sample ofbiomolecular analyte is analyzed without out having to remove the testmodule 55 from the holder 20 to clean and prepare the test module 55 fora second run. As a result of adding the second sample arm 112 b, asignificant savings in processing time can be achieved.

To analyze a sample, various electrophoresis procedures can be used toprepare and separate the sample into STRs. One of these proceduresinvolves heating the test module 55 to a temperature of about 40 toabout 70 degrees C. while applying a voltage to separate thebiomolecular analyte. For example, in one preferred embodiment, thetemperature of the test module is held at 50 degrees C. duringelectrophoresis. Prior to applying separation conditions to the testmodule 55, at least one biasing condition is applied to each of themicrochannels 110 in the test module 55 to prepare the sample forseparation. A first biasing condition moves the buffer solution into theseparation channel 116 from the cathode opening 118 a towards the anodeopening 118 b. The first biasing condition is represented in FIG. 6, inwhich a voltage of less than 10 KV is applied between the cathode 102and the anode 100. A second biasing condition shown in FIG. 7 applies avoltage between the sample arm 112 and the waste arm 114, and moves theanalyte from the sample arm 112 towards the waste arm 114. A thirdbiasing condition shown in FIG. 8 applies a voltage from the waste arm114 to the sample arm 112 to move the analyte from the waste arm 114towards the sample arm 112. A fourth biasing condition shown in FIG. 9applies a voltage between the cathode 102 and the waste arm 114 to movethe analyte away from the separation channel and back into the samplearm 112 and the waste arm 114. The first biasing condition is appliedwith a first power supply and the second, third, and fourth biasingconditions are applied with a second power supply. When both the firstbiasing condition and one of the second, third, or fourth biasingconditions are applied at the same time, further biasing conditions canbe applied to the microchannel 110. For example, as shown in FIG. 10, afifth biasing condition, which is a combination of the first and secondbiasing conditions, moves the analyte from the sample arm 114 towardsthe waste arm 114, while also moving the analyte in the separationchannel 116 from the cathode 102 towards the anode 100. A sixth biasingcondition is formed by the combination of the first and third biasingconditions and is shown in FIG. 11. A seventh biasing condition, shownin FIG. 12, is a combination of the first and fourth biasing conditions.The seventh biasing condition moves the analyte in the area ofintersection between the separation channel and the sample and wastearms (e.g., intersection region 125) towards the sample and waste arms112 and 114, while also moving the analyte in remaining or otherportions of the separation channel 116 from the cathode 102 towards theanode 100.

Sequential application of the above biasing conditions allows for anumber of different functions including loading, stacking, separation,and prevention of excess analyte from diffusing into the separationchannel during separation. Electric fields of between about 50 to about500 V/cm are typically applied across the cathode 102 and the anode 100,while electric fields of about 50 V/cm to about 500 V/cm are applied tothe waste and sample arm openings 118 d and 118 c. Loading of theanalyte into the separation channel 116 is accomplished by applying thesecond bias configuration together with an electric field of about 50V/cm to about 400 V/cm for 0.5 to 5 minutes about the sample and wastearms 112 and 114. Stacking of the analyte at the intersection region 125is accomplished by applying the first bias configuration together withan electric field about the channels 110 of up to about 500 V/cm for arelatively short time frame (e.g., about 1 second to about 10 seconds).The conductivity difference between the intersection region 125 and theseparation channel 116 forces the analyte in the intersection region 125to form a compressed band, which allows for high resolution separations.Separating the analyte is accomplished by applying the first biasingconfiguration together with an electric field of about 50 to about 500V/cm to move the compressed band of analyte towards the anode 100 fromthe cathode 102. The first biasing configuration is applied to thecompressed bands until the largest fragments of interest have movedthrough a detection zone 150 (i.e., a portion of the separation channelwhich interacts with the laser beam). Analyte from the sample arm 112and waste arm 114 can diffuse into the separation channel 116 leading todistortions in the electrophoregram baseline and a reduction in a signalto noise ratio. As a result, excess analyte in the sample arm 112 orwaste arm 114 is prevented from entering the separation channel 116during analysis by applying the seventh biasing configuration to ensurethat a negative electric field is setup between the cathode opening 118a and each of the sample and waste openings 118 d and 118 c,respectively. In some embodiments of the invention, the seventh biasingconfiguration as described above is applied for the remainder of ananalysis to ensure the quality of the signal collected by thefluorescence excitation and detection system 40. In other embodiments,once the conduction bands have moved away from the intersection region125 towards the detection zone 150, the seventh biasing condition isswitched off and the first biasing condition is applied. In thisembodiment, excess analyte is unable to diffuse into the separationchannel 116 because the conduction bands have moved away from theintersection region 125. As a result, excess analyte cannot interferewith data collection. In another embodiment, excess analyte is preventedfrom entering the separation channel 116 by removing analyte from thesample opening 118 d and/or waste opening 118 c during separation.Either the third or the fourth biasing configuration can be appliedduring separation to move excess analyte to either the sample arm 112 ora combination of the sample and waste arms 114 for removal.

Once the biomolecular analyte sample is separated, biasingconfigurations are applied to move the separated components (STR loci)towards the detection zone 150. The laser beam emitted from the laser 60scans through the detection zone 150 and the induced fluorescent lightfrom the DNA is collected and transmitted by the light detectors 64within the fluorescence excitation and detection system 40. As the laserbeam scans through the detection zone 150, laser light is absorbed bythe fluorescently tagged STR loci that are moving therethrough. Theinduced fluorescence from each of the tagged STR is collected by thefluorescence excitation and detection system 40 and transmitted to thedetectors 64. Through a combination of dichroic mirrors and band-passfilters, the emission wavelength of the specific fluorescently taggedand hence the specific STRs are identified. These results can becompared to industry standards or other samples for forensicidentification purposes.

In the present invention, a novel “lanefinding” process is used toautomatically compensate for changes in chip position or othermisaligned elements in the optical train without requiring the user toperform a realignment procedure. In some embodiments, “lanefinding” isincluded as one of the components used to ruggedized apparatus describedherein.

Alignment issues between the fluorescence excitation and detectionsystem 40 and the test module 55 can introduce degradation in signalquality, specifically the relative intensity level of the inducedfluorescence relative to a background noise level. “Lanefinding” isprocess used in some embodiments of the present invention to maximizethe signal to noise ratio of the fluorescence data from the STRs and tomore accurately find the detection zone 150. Specifically, if thealignment between the excitation energy source and the channels withinthe test modules 55 is poor, tagged DNA moving through the detectionzone 150 will not be efficiently excited. In addition, other regions ofthe chip, such as, for example, from between the channels, will beexcited and thus will result in the generation of excessive backgroundfluorescence. Misalignment between the excitation energy beam and thechannel will also result in the collection of excessive backgroundfluorescence relative to the reduced induced fluorescence from the DNA,thereby reducing the signal to noise ratio. As a result of sampling datafrom other regions other than the detection zone 150, unreliable and/orunusable results are produced. The alignment between the fluorescenceexcitation energy beam and the detection zone can be monitored andcorrected by an attached processor, such as an attached computer runningsoftware with the lanefinding program running thereon.

Unlike prior art devices, the devices of the present invention aredesigned to incorporate removeable test modules 55 or chips. In thepresent invention, some embodiments use novel lanefinding methods todetermine the location of each microchannel 110 after initial insertionor removal and re-insertion of a test module 55. Lanefinding eliminatesthe need for manual re-alignment of the plurality of optics within thefluorescence excitation and detection system 40 when test modules 55 areremoved and/or reinserted. In one embodiment, lanefinding removesspurious peaks introduced from aberrations, including withoutlimitation, aberrations caused by scratches or other imperfections inthe transparent test module 55, and the presence of foreign particles inthe transparent test module 55 and the like. Referring to FIG. 13, theexcitation energy source (e.g., laser beam) is scanned through thedetection zone 150 on a particular test module before using device 10 toanalyze a sample. As the laser beam passes through each channel 110 itis deflected by index of refraction discontinuities between thetransparent material between the channels 110 and the medium within thechannels. The deflected light beam can scatter and/or waveguide throughthe test module 55 and can be detected by photodiodes 170, 172 placed oneither side of test module. Referring to FIG. 14, there are four generalcases where laser light incident on the channels is deflected towardsand detected by the photodiodes. As shown in FIG. 14, laser path oneintercepts a front edge of a channel and is deflected towards photodiode170. Laser path two does not intercept a channel, but rather travelsonly through the transparent material of the test module. As a result,no light is directed towards either photodiode 170 or 172. Laser paththree intercepts the center or middle of a microchannel. As shown inFIG. 14, laser beam in laser path three is directed towards bothphotodiode 170 and 172 and is thus the best location to detect data.Finally, laser path four intercepts a back edge of a channel and isreflected towards photodiode 172 only. FIG. 15 shows a typical waveformat photodiode 172 as the laser beam scans across one microchannel. Thewaveform shows that the front edge position of the channel 110 islocated at about 1385 counts, the middle position is located at about1425 counts, and the back edge of the channel 110 is located at 1475counts.

In one embodiment, the lanefinding software determines the position ofthe middle of each channel 110 by a series of steps designed toeliminate spurious peaks and to identify the front and back edgeposition of each microchannel 110. In this embodiment, the intensitiesof the peaks from all of the channels detected by the photodiodes 170and 172 is normalized to a maximum value of one. At least three scans ofthe detection zone are recorded and a three-point boxcar average isperformed to smooth out the collected data and to smooth out the tracesto ensure interpretation accuracy. All peaks within the detection zone150 are identified and any peak not identified on both waveformscollected by photodiode 170 and photodiode 172 is eliminated. Theremaining peaks are reviewed for spurious peaks introduced byaberrations, such as, for example, aberrations caused by scratches orother imperfections or foreign particles in the transparent test module55. The peaks resulting from these flaws or scratches are identified byexamining the space between each peak. As shown in FIG. 15, each of thepeaks associated with a particular channel are spaced at regularintervals corresponding to the front, middle and back positions of thechannel. That is, each peak is located within a one-third channel widthdistance apart from each other. Thus, any peak that is not correlated toother peaks with a one-third channel width distance there between iseliminated from the waveform. All remaining peaks are used to determinethe middle position of each channel 110.

In one embodiment, the middle position of the channels are determined byfirst identifying the first peak in the waveform. This peak isidentified as the front edge of the first channel. Peaks within 1.2channel widths from the front edge are identified. These identifiedpeaks are averaged to determine the middle position of the firstchannel. The next peak located along the waveform at a distance greaterthan 1.2 channel widths from the first peak is identified and labeledthe front edge of the second channel. Peaks within 1.2 channel widthsfrom the front edge of the second channel are identified and averagedtogether to determine a middle position of the second channel. Thisprocess of identifying the front edge and middle position is continueduntil all channels with the test module 55 are identified. The middlepositions of each channel are averaged together to determine an averageoffset from the front edge position of the channels. This offset isrepresentative of the distance between the front edge and the middleposition of each channel. A reference file including the front edgepositions of each of the channels is updated with the determined offsetto guide the motion of the scanner 62 to direct the laser beam to theappropriate positions for data collection.

In certain embodiments of the invention, the ruggedized nucleic acidseparation/sequencing device 10 further includes a novel system forremoving background noise from the signal collected by the fluorescenceexcitation and detection system 40. Specifically, the fluorescencesignal that is collected by the detection system and transmitted to thedetectors consists predominantly of two sources, tagged bioanalyte andbackground. The background component relates to all detectablecomponents except for the tagged bioanalyte and includes fluorescence ofthe test module 55, any fluorescing elements in the detection path, andbackground light that is not blocked. This background is of a fixedintensity level and directly adds to the signal from the taggedbioanalyte. In one embodiment of the present invention, baselinesubtraction circuitry is implemented to remove the fixed backgroundlevel from the detector prior to electronic amplification and conversionof the signal from analog to digital form. The removal of backgroundnoise (i.e., background offset) allows for a larger dynamic range forsignal detection. In prior art systems, detectors convert detectedfluorescence (background and signal) into a current, which is thenconverted into a voltage through electronic amplification. The analogvoltage is converted to digital form through an analog to digitalconverted. In certain embodiments of the present invention, a currentsource is connected to the detector circuitry, directly after thedetector and before electronic amplification, to enable the applicationof a subtractive current for background removal and leading to a largerdynamic range than prior art devices. In the present invention, thecurrent is controlled electronically and is user selectable.

One of the advantages of the background subtraction system of thepresent invention over prior art systems is increased signal dynamicrange. As a result of the increased signal dynamic range, a larger rangeof sample concentrations can be detected. For example, a samplecollected from a crime scene typically includes a large concentration ofa victim's DNA and a small concentration of a perpetrator's DNA. Thatis, the sample includes a dilute amount of the perpetrator's DNA and ahigh concentration of the victim's DNA. The background subtractionsystem of the present invention allows a user to detect a large range ofsignal/sample concentrations within a single sample without saturatingan A/D converter with background noise, while having enough sensitivityto detect the dilute concentration of a second source of DNA (e.g.,perpetrator's DNA).

EXAMPLES

The following examples are provided to further illustrate and tofacilitate the understanding of the invention. These specific examplesare intended to be illustrative of the invention and are not intended tobe limiting.

Example 1

The following example illustrates a forensic use of a ruggedized nucleicacid analysis device in accordance with the present invention. In thisexample, a ruggedized nucleic acid analysis apparatus similar to the oneshown in FIGS. 1A, 1B, and 1C was used to analyze a reference sample.

The reference sample consisted of an allelic ladder from a commerciallyavailable STR kit (AMPFISTR® SGM PLUS®, from Applied Biosystems, FosterCity, Calif.) and size standard (GENESCAN™ 400HD (ROX™) Size Standard,from Applied Biosystems, Foster City, Calif.). The sample was preparedusing 2 microliters of allelic ladder, 0.5 microliters of size standard,and 10.5 microliters of deionized water. The sample was denatured priorto analysis by heating the sample to about 90 degrees C. for about 3minutes followed by rapidly cooling the sample on ice. The sample wasthen injected into a cleaned microfluidic chip.

Prior to sample injection, the microfluidic chip was cleaned andconditioned to eliminate excess ions in the channels of the chip.Specifically, each channel in the microfluidic chip was filled through apress with 4% LPA sieving material (Dakota Scientific, Sioux Falls,S.D.) to clean the channels and the anode, cathode, sample arm, andwaste arm openings. After cleaning was completed, the anode and cathodeopenings were filled with 500 microliters of 1×TTE buffer (availablefrom Dakota Scientific, Sioux Falls, S.D.), the waste arm opening wasfilled with 33 microliters of 1×TTE buffer, and the sample arm openingwas filled with 13 microliters of deionized water. Each of the channelswas conditioned through a preelectrophoresis process in which a 190 V/cmelectric field was applied across the cathode and anode for 6 minutesfollowed by a 875 V/cm electric field was applied across the sample armand waste arm for 3 minutes.

Following preelectrophoresis, the cathode, anode, sample arm, and wastearm openings were cleaned and then filled. The cathode and anodeopenings were filed with 500 microliters of 1×TTE buffer, the waste armopening was filled with 33 microliters of TTE buffer, and the sample armopening was filled with 13 microliters of the sample as described above.

The sample was loaded into the separation channel by applying a field of875 V/cm across the sample and waste arms while simultaneously applyinga field of 88 V/cm across the anode and cathode for 1.5 minutes.Following loading, the sample was separated and excess sample was pulledback away from the separation channel by applying 190V across thecathode and anode while simultaneously applying 800 V to each of thesample arm and waste arm openings. These conditions were applied to themicrofluidic chip for 45 minutes to separate the sample.

The fluorescence excitation and detection system was then activated toexcite fluorescently tagged STR loci within the separated sample. FIGS.16A, 16B, 16C, and 16D show the raw electrophoregrams of the allelicladder generated by the data collected by the fluorescence excitationand detection system. Each of the electrophoregrams collected representone intensity data captured by one of the photomultiplier tubes locatedwithin the apparatus. That is FIG. 16A shows the data collected by thephotomultiplier tube configured to amplify and detect blue wavelengthlight, FIG. 16B shows the data collected by the photomultiplier tubeconfigured to amplify and detect green wavelength light, FIG. 16C showsdata collected by the photomultiplier tube configured to amplify anddetect yellow wavelength light, and FIG. 16D shows data collected by thephotomultiplier tube configured to amplify and detect red wavelengthlight.

The data from each of the electrophoregrams was corrected using signalprocessing methods for baseline smoothing, subtraction, and colorcorrection with a 4×4 matrix method as described by L. Li et al. inElectrophoresis 1999, volume 20, issue 1, pages 1433-1442, thedisclosure of which is hereby incorporated by reference in its entirety.FIGS. 17A, 17B, 17C, and 17D show expanded views of the corrected data.FIG. 17A, which shows the corrected trace of the data for the bluewavelength light, shows the presence of the following loci: D3S1358,VWA, D16S539 and D2S1338 and all of the alleles (i.e., 8, 14, 9, and 14,respectively) associated with each of the loci. In FIG. 17B, thecorrected trace of green wavelength light data shows the presence ofAmelogenin and loci D8S1179, D21S11, D18S51 and all alleles associatethereto. FIG. 17C shows the corrected trace of yellow wavelength lightdata. FIG. 17C shows the presence of the following loci: D19S433, TH01,and FGA (both low and high molecular weight sets) together with allalleles associated with each loci. FIG. 17D shows the corrected trace ofred wavelength light data. The corrected red wavelength light traceshows size standard peaks at 90, 100, 120, 150, 160, 180, 190, 200, 220,240, 260, 280, 290, 300, 320, 340, 360, 380, and 400.

Clear identification of all 11 loci associated alleles together with allof the size standards in the sample show that the ruggedized nucleicacid analyzing apparatus has a discrimination ability well suited forforensic analysis. Specifically, the results shown in FIGS. 16A-D andFIGS. 17A-D show that this apparatus has a discrimination power of about1 to 3.3×10¹².

Example 2

The following example illustrates a DNA sequencing use of a ruggedizednucleic acid analysis device in accordance with the present invention.In this example, a ruggedized nucleic acid analysis device similar tothe one shown in FIGS. 1A, 1B, and 1C was used to analyze a sampleincluding a DNA template from an HIV amplicon B.FR.HXB2, with primer GB107. The sample was amplified and labeled with a commercially availablecycle sequencing kit (Thermo Sequenase II Dye Terminator CycleSequencing Premix Kit) available from Amersham Biosciences, now part ofGE Healthcare (Waukesha, Wis.). A cycle sequencing reaction wasperformed following the manufacturer's recommended procedure. The totalsequencing reaction consisted of 750 nanograms of DNA template, 1microliter of 5 micromolar primer, two microliters of Thermo SequenaseII reagent mix A, 2 microliters of Thermo Sequenase II reagent mix B,and water to bring the total volume to 20 microliters. The reaction wascycled with the following program, with steps 2 through 4 being repeated30 times: 1) 96 degrees C., 1 minute; 2) 96 degrees C., 30 seconds; 3)50 degrees C., 15 seconds; 4) 60 degrees C., 1.5 minutes; and 5) 60degrees C., 5 minutes. The reaction product was stored at 6 degrees C.until ready for use at which time the sample is resuspended in 130microliters of water and denatured by heating the sample to 70 degreesC. for 3 minutes followed by cooling on ice.

Prior to injecting the sample into the transparent test module thechannels of the test module were cleaned and conditioned (i.e.,subjected to preelectrophoresis) as described in Example 1. Followingpreelectrophoresis, the anode, cathode, sample arm, and waste armopenings of the each channel were cleaned and filled. The cathode andanode openings were filled with 500 microliters of 1×TTE bufferavailable from Dakota Scientific, Sioux Falls, S.D. The waste armopening was filled with 33 microliters of 1×TTE buffer, and the samplearm opening was filed with 13 microliters of the resuspended anddenatured sample.

The sample was loaded into the separation channel from the sample armopening by applying a field of 875 V/cm across the sample arm and wastearm for 60 seconds. The sample was then separated with excess samplebeing pushed back into the sample and waste arm openings by applying 190V across the cathode and anode, while simultaneously applying 400 V toeach of the sample and waste arm openings. These voltage conditions wereapplied for 60 minutes to separate the sample

The fluorescence excitation and detection system was then activated toexcite fluorescently tagged DNA within the separated sample. FIGS. 18A,18B, 18C, and 18D show the raw electrophoregrams of the DNA sequencegenerated by the data collected by the fluorescence excitation anddetection system. Each of the electrophoregrams collected represent oneintensity data captured by one of the photomultiplier tubes locatedwithin the apparatus. That is FIG. 18A shows the data collected by thephotomultiplier tube configured to amplify and detect blue wavelengthlight, FIG. 18B shows the data collected by the photomultiplier tubeconfigured to amplify and detect green wavelength light, FIG. 18C showsdata collected by the photomultiplier tube configured to amplify anddetect yellow wavelength light, and FIG. 18D shows data collected by thephotomultiplier tube configured to amplify and detect red wavelengthlight.

The data from each of the electrophoregrams was corrected using signalprocessing methods as described by L. Li et al. in Electrophoresis 1999,volume 20, issue 1, pages 1433-1442. Further processing of the correctedtraces was accomplished by base calling, which associates one of thefour nucleotides with each peak in the trace. The traces are smoothed byusing a 9-point boxcar average and then the traces are differentiated.Peaks are identified by evaluating the differentiated traces to locatezero crossing with a positive to negative slope change. Peaks identifiedin the blue wavelength light, green wavelength light, yellow wavelengthlight and red wavelength light traces are correlated with bases G, A, T,and C, respectively. One skilled in the art of data processing canappreciate the rudimentary nature of this base calling routine, whichwas used to demonstrate the effectivity of this device. Moresophisticated base callers will typically generate longer contiguousreads with the same data as compared to the above described method. Adetailed description of base calling can be found at “Base-Calling ofAutomated Sequencer Traces Using Phred I. Accuracy Assessment” by Ewinget al. in Genome Research, 1998, volume 8, pages 175-185 and“Base-Calling of Automated Sequencer Traces Using Phred II. ErrorProbabilities” by Ewing et al., in Genome Research, 1998 volume 8, pages186-194, the disclosures of which are hereby incorporated by referencein their entirety.

FIGS. 19A, 19B, 19C, and 19D show expanded views of the corrected data.FIG. 19A, which shows the corrected trace of the data for the bluewavelength light, shows the clear identification of the nucleotideguanine. In FIG. 19B, the corrected trace of green wavelength light datashows the clear identification of adenine. FIG. 19C shows the correctedtrace of yellow wavelength light data. FIG. 19C shows the clearidentification of thymine. FIG. 19D shows the corrected trace of redwavelength light data. The corrected red wavelength light trace showsthe identification of cytosine.

Referring to FIGS. 20A, 20B, and 20C, clear identification of thenucleotides associated with the DNA template from the known HIV ampliconB.FR.HXB2 show that the ruggedized nucleic acid analyzing device is wellsuited for sequencing analysis to be performed at point of carelocations. FIG. 20A (i.e., FIG. 20A-1 and FIG. 20A-2) shows a sequencelisting of the known amplicon. FIG. 20B shows a sequence listingobtained from analysis of the results generated from the ruggedizednucleic acid analyzing device used in this example. The sequence listingshown in FIG. 20B is unedited and was generated after the data as shownin FIGS. 19A-19D was reverse-complemented using techniques known in theart due to the reverse primer used with this sample. The uneditedsequence listing shown in FIG. 20B consists of 378 contiguous bases thatexhibit 100% identification with the amplicon of FIG. 20A, from bases436 to 813. This result is further improved by manually editing the datato remove and replace obvious mistakes incorporated during base calling.FIG. 20C shows the sequence listing obtained after manually editing thedata. The edited sequence listing consists of 544 contiguous bases thatexhibited 100% identification with the amplicon from bases 297 to 840.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the claims.

For example, while the present invention in certain embodiments has beendescribed as using a transparent test module including one or moremicrochannels disposed therein, other types of channels can be used.Specifically, the transparent test modules can include channels sized inaccordance with standard non-microfluidic capillary electrophoresisprocedures. As yet another example, while the present invention has beendescribed as using particular lanefinding scheme to determine thelocation of the middle of each channel, other lanefinding algorithms canbe applied. In certain embodiments of the invention, the lane findingscheme can be modified to detect changes in fluorescence rather thanlight intensity. In this particular embodiment, photomultiplier tubesare used to capture the reflected signal rather than photodiodes.

What is claimed is:
 1. A system for finding the position of separationchannels in an electrophoresis system comprising an electrophoresis chipand an instrument capable of separating and detecting of DNA fragmentslabeled with at least one fluorescent dye comprising: (a) a removableelectrophoresis chip comprising: a substrate having top and bottomsurfaces, further comprising an anode portion, a cathode portion, and aseparation channel between the anode and cathode portions, and havingtop and bottom surfaces such that the top surface of said substratelayer is bonded with the bottom surface of said cover layer to form atleast one microfluidic channel, a first via located at said anode and influidic communication with at least one of the microfluidic channels, asecond via located at said cathode and in fluidic communication with atleast one of the microfluidic channels, a detection zone located in theseparation channel; and (b) an electrophoresis instrument capable ofreceiving said chip, comprising, (i) a processor, (ii) an opticalsystem, said optical system comprising said fluorescence excitationenergy beam, and at least one photodiode, positioned on a first side ofsaid chip when said chip is inserted into said instrument, and at leastone light detector, and, (iii) an electrical system for delivering saidbeam to said detection zone, and having an anode and a cathode, saidanode in contact with said anode portion of said substrate, and saidcathode in contact with said cathode portion of said substrate, andwhereby the processor directs the beam to pass through the detectionzone, and the scattered light from the separation channel is detected bysaid photodiode, thereafter, said processor finds and stores theposition of the separation channel to detect data, so that when saidelectrical system delivers a voltage from said cathode to said anode,moving said labeled DNA fragments from said cathode portion into saidseparation portion within the detection zone; said processor uses saidposition to measure fluorescent signal using said light detector.
 2. Thesystem of claim one wherein a second photodiode is positioned on asecond side of said chip opposite said first side.
 3. System of any ofclaim 1 or 2 wherein the processor further comprises baselinesubtraction circuitry to remove the fixed background level from thedetector prior to electronic amplification and conversion of the signalfrom analog to digital form.4. A method of determining at least onecenter channel location in a transparent test module including aplurality of separate fluid channels disposed therein, the methodcomprising: a) providing a transparent test module including theplurality of separate fluid channels, each of the separation channelshaving a front end position, a center channel position, and a knownchannel width; b) scanning over a portion of the transparent test moduleincluding each of the plurality of separation channels with a light beamto generate a reflected light beam; c) collecting the reflected lightbeam with a light detector to generate a waveform of intensity throughthe portion of the transparent test module including each of theplurality of separation channels; d) eliminating peaks determined not tobe associated with the plurality of separation channels; e) identifyinga location within the waveform of the front end position for each of theplurality of separation channels; f) identifying all remaining peaksalong the waveform within a first distance extending from each of theidentified front end positions; and g) determining the center channelpositions of each of the plurality of separate fluid channels from anaverage of locations of all of the remaining peaks within the firstdistance from each of the identified front end positions.
 4. A method ofdetermining at least one center channel location in a transparent testmodule including a plurality of separate fluid channels disposedtherein, the method comprising: a) providing a transparent test moduleincluding the plurality of separate fluid channels, each of theseparation channels having a front end position, a center channelposition, and a known channel width; b) scanning over a portion of thetransparent test module including each of the plurality of separationchannels with a light beam to generate a reflected light beam; c)collecting the fluorescence with a light detector to generate a waveformof intensity through the portion of the transparent test moduleincluding each of the plurality of separation channels; d) eliminatingpeaks determined not to be associated with the plurality of separationchannels; e) identifying a location within the waveform of the front endposition for each of the plurality of separation channels; f)identifying all remaining peaks along the waveform within a firstdistance extending from each of the identified front end positions; andg) determining the center channel positions of each of the plurality ofseparate fluid channels from an average of locations of all of theremaining peaks within the first distance from each of the identifiedfront end positions.